琼脂糖凝胶电泳1

琼脂糖凝胶电泳1 DNA琼脂糖凝胶电泳 跑胶即走电泳，是DNA 和protein 最基本的定性定量方法。一般是琼脂糖胶或page胶，琼脂糖胶一般是检测DNA的，可以检验一下你到底提到dna没过确定你提dna分子量的大小:page胶一般是检测蛋白特性的，通过page胶里marker的分子量大小来确定你需要的目的蛋白分子量大小，如果有杂带那就可以判断那条带是你需要的目的条带，如果想知道蛋白浓度，还可以在page胶里带上一条定量marker，通过条带粗细，来判断你需要蛋白的浓度。二者原理一致，小分子量用大浓度的胶，大分子量用小浓度的胶，特别小的DNA用丙烯酰胺凝胶。 一、 基本原理 当把一个带净电荷(q)的颗粒放入电场，便有一个电场力(F)作用于其 上。F的大小取决于颗粒静电荷量及其所处的电场强度(E)，它们之间的关系 可以表示成:F=E×q。 由于F的作用，使带电颗粒在电场中向一定方向泳动。此颗粒在泳动的过程 中还受到一个相反方向的摩擦力(f*v)阻挡。当这两种力相等时，颗粒则以 速度(v)向前泳动:v=F/f,其中f为摩擦系数。根据Stoke公式，阻力大小 取决于带电颗粒的大小、形状以及所处介质的粘度，即f=6πγη, γ为颗 粒半径，η为介质粘度。该公式指球形颗粒所受的阻力。代入F,E×q得到: q/6πγη.从这个公式可以看出，带电颗粒在电场中泳动的速度与电v=E× 场强度和带电颗粒的净电荷量成正比，与颗粒半径和介质粘度成反比。 带电颗粒在电场中泳动的速度常用泳动度(m)或者迁移率以下列公式表示: m,μ/E=d*l/V*t d为带电颗粒泳动的距离(cm)，l为支持物的有效长度(cm)，t为通电时 间(s)，V为加在支持物两端的电压。 在一定条件下，任何带电颗粒都具有自己特定的泳动度。它是胶体颗粒 的一个物理常数，可用其鉴定蛋白质、核酸等物质的纯度，还可以用其来研 究蛋白质、核酸等物质的一些理化性质。影响泳动度的因子有颗粒的性质、 电场强度、溶液的性质等。 二、 琼脂糖凝胶电泳 通常情况下，核酸类物质的分离、鉴定是采用琼脂糖凝胶(做支持物)电 泳法进行的。用琼脂糖分离线性DNA时，其迁移率与该物质分子质量的关系 密切，而与结构和碱基组成无关。这也是采用凝胶电泳法测定核酸分子质量 的依据所在。此方法除了可以分离线性DNA外，还可以分离、细菌质粒 的闭环DNA和开环DNA，以及分子质量不等的RNA片段。一般实验室采用的 琼脂糖凝胶的浓度为0.3%——2%。不同的凝胶浓度，可以分离不同长度的 DNA片段。具体见表格: 琼脂糖凝胶 /% m/V 分离线性DNA片段的范围 /kb 0.3 50---60 0.6 1----20 0.7 0.8---10 0.9 0.5---7.0 1.2 0.4----6.0 1.5 0.3---3.0 1 2.0 0.1---2.0 琼脂糖凝胶电泳常用的缓冲液: 缓冲液 pH 1.50mmol/L Tris-NaHPO-1--5mmol/L EDTA 7.5---8.0 24 2.50mmol/L NaHPO- NaHPO-1--5mmol/L EDTA 7.5---8.0 2424 3.50mmol/L Tris-乙酸-1--5mmol/L EDTA 7.5---8.0 4.50mmol/L 乙酸钠-乙酸-1--5mmol/L EDTA 7.5---8.0 5.89mmol/L Tris-HBO-1--5mmol/L EDTA 8.3 33 琼脂糖凝胶电泳常用的样品缓冲液(示踪染料):0.25%溴酚蓝-0.25%二甲苯 青FF-30%甘油水溶液，并且这两种指示剂在琼脂糖凝胶中的迁移率不同，可 以指示一定片段长度的DNA迁移率(具体见下表)。 琼脂糖凝胶浓度 溴酚蓝 二甲苯青 0.6% 1kb / 1.0% 0.6kb 2kb 1.4% 0.2kb 1.6kb 2.0% 0.15kb / 核酸分子量标准(Marker):DNA电泳一定要用DNA Marker或者是已知大小的正对照DNA来估计片段的大小。应该选择在目标片段大小附近Ladder 较密的Marker，这样对于目标片段大小的估计才比较准确。 核酸电泳染色剂:核酸电泳以后，需要经过染色才能显现带型，最常用的染色剂是溴化乙锭(EB)，其次是银染法。 1、溴化乙锭(ethidium bromide): 一种荧光染料，可以嵌入核酸的双链碱基对 之间，在紫外光线的激发下，发出红色荧光。 2、银染法:染色液中的银离子可与核酸形成稳定的复合物，然后用还原剂如甲 醛使银离子还原成银颗粒，可以将核酸带染成黑色。灵敏度比EB高，但是 DNA不易回收。 琼脂糖凝胶电泳的操作步骤如下: 1. 制备1%琼脂糖凝胶(大胶用70ml,小胶用50ml):称取0.7 g(0.5 g)琼脂糖置于锥形瓶中,加入70 ml(50ml)1×TAE,瓶口倒扣小烧杯.微波炉加热煮沸3次至琼脂糖全部融化,摇匀,即成1.0%琼脂糖凝胶液. 2. 胶板制备:取电泳槽内的有机玻璃内槽(制胶槽)洗干净,晾干,放入制胶玻璃板.取透明胶带将玻璃板与内槽两端边缘封好,形成模子.将内槽置于水平位置,并在固定位置放好梳子.将冷却到65?左右的琼脂糖凝胶液混匀小心地倒入内槽玻璃板上,使胶液缓慢展开,直到整个玻璃板表面形成均匀胶层.室温下静置直至凝胶完全凝固,垂直轻拔梳子,取下胶带,将凝胶及内槽放入电泳槽中.添加 1×TAE电泳缓冲液至没过胶板为止. 3. 加样:在点样板或parafilm上混合DNA样品和上样缓冲液,上样缓冲液的最终稀释倍数应不小于1X.用10 ul微量移液器分别将样品加入胶板的样品小 2 槽内,每加完一个样品,应更换一个加样头,以防污染,加样时勿碰坏样品孔周围的凝胶面.(注意:加样前要先记下加样的顺序). 4. 电泳:加样后的凝胶板立即通电进行电泳,电压60-100V,样品由负极(黑色)向正极(红色)方向移动.电压升高,琼脂糖凝胶的有效分离范围降低.当溴酚蓝移动到距离胶板下沿约1cm处时,停止电泳. (5)电泳完毕后,取出凝胶,用含有0.5 ug/ml的溴化乙锭1×TAE溶液染色约20 min,再用清水漂洗10 min. (6)观察照相:在紫外灯下观察,DNA存在则显示出红色荧光条带,采用凝胶成像系统拍照保存. 琼脂糖凝胶电泳 一,实验原理 琼脂糖凝胶电泳是分离和纯化DNA 片段的常用技术.把DNA样品加入到一块包含电解质的多孔支持介质(琼脂糖凝胶)的样品孔中,并置于静电场上.由于DNA分子的双螺旋骨架两侧带有含负电荷的磷酸根残基,因此在电场中向正极移动.在一定的电场强度下,DNA分子的迁移速度取决于分子筛效应.具有不同的相对分子质量的DNA片段泳动速度不一样,因而可依据DNA分子的大小来使其分离.凝胶电泳不仅可分离不同分子质量的DNA, 也可以分离相对分子质量相同,而构型不同的DNA分子.在电泳过程中可以通过示踪染料或相对分子质量标准参照物和样品一起进行电泳而得到检测.相对分子质量标准参照物相对可以提供一个用于确定DNA片段大小的标准.在凝胶中加入少量溴化乙锭(ethidium bromide, EB),其分子可插入DNA的碱基之间,形成一种络合物,在254,365nm波长紫外光照射下,呈桔红色荧光,因此也可对分离的DNA进行检测. 一般琼脂糖凝胶电泳适用于大小在0.2kb,50kb范围内的DNA片段.本实验介绍琼脂糖凝胶的制备以及琼脂糖凝胶电泳在DNA片段分离中的应用方法. 琼脂糖凝胶浓度(%) 线状DNA分子分离范围(kb) 0.3 5--60 0.6 1--20 0.9 0.5--7 1.2 0.4--6 1.5 0.2--3 2.0 0.1--2 3 琼脂糖凝胶电泳是用于分离纯化和鉴定核酸的方法，根据琼脂糖的溶解温度，把琼脂糖分为一般琼脂糖和低熔点琼脂糖。低熔点琼脂糖的熔点为62--65,溶解后在37下维持液体状态约数小时,主要用于DNA片断的回收、质粒与外源性DNA的快速连接等。 DNA在琼脂糖凝胶中的迁移速度与琼脂糖浓度、DNA分子量及其构象、电泳缓冲液、电场强度等因素有关，一般说来，DNA片断越大或者琼脂糖浓度越大，其迁移速率越大;而电场强度越高，其迁移速率越大。不同浓度琼脂糖凝胶DNA分离范围见上图表。 二,仪器及试剂 1.仪器及耗材: 水平电泳槽,电泳仪,凝胶成像分析系统,微波炉,微量移液器,透明胶带,点样或parafilm,100 ml或250 ml锥形瓶,量筒,吸头等. 2.试剂及配制: 50×TAE缓冲液的配制:2 mol/L Tris-乙酸,0.05 mol/L EDTA(pH 8.0) 配制1000 ml Tris 242 g 冰乙酸 57.1 ml 0.5 mol/L EDTA 100 ml 加入600 ml去离子水后搅拌溶解,将溶液定容至1 L后.高温高压灭菌,室温保存. 1×TAE缓冲液的配制: 称量20 ml的50×TAE缓冲液,再加入980 ml的去离子水. 溴化乙啶贮存液:10 mg/ml 溴化乙啶 配制:100 ml 称取1 g溴化乙啶,置于100 ml烧杯中,加入80 ml去离子水后搅拌溶解.将溶液定容至100 ml后,转移到棕色瓶中.室温保存. 6×上样缓冲液:0.25%溴酚蓝,0.25%二甲苯青FF,30%甘油. 配制:10 ml 溴酚蓝 25 mg 4 二甲苯青FF 25 mg 甘油 3 ml 用6×TAE缓冲液定溶至10 ml,分装成1 ml/管.-20?保存. 其它试剂:DNA样品,DNA Ladder ,琼脂糖, 三,操作步骤 1. 制备1%琼脂糖凝胶(大胶用70ml,小胶用50ml):称取0.7 g(0.5 g)琼脂糖置于锥形瓶中,加入70 ml(50ml)1×TAE,瓶口倒扣小烧杯.微波炉加热煮沸3次至琼脂糖全部融化,摇匀,即成1.0%琼脂糖凝胶液. 2. 胶板制备:取电泳槽内的有机玻璃内槽(制胶槽)洗干净,晾干,放入制胶玻璃板.取透明胶带将玻璃板与内槽两端边缘封好,形成模子.将内槽置于水平位置,并在固定位置放好梳子.将冷却到65?左右的琼脂糖凝胶液混匀小心地倒入内槽玻璃板上,使胶液缓慢展开,直到整个玻璃板表面形成均匀胶层.室温下静置直至凝胶完全凝固,垂直轻拔梳子,取下胶带,将凝胶及内槽放入电泳槽中.添加 1×TAE电泳缓冲液至没过胶板为止. 3. 加样:在点样板或parafilm上混合DNA样品和上样缓冲液,上样缓冲液的最终稀释倍数应不小于1X.用10 ul微量移液器分别将样品加入胶板的样品小槽内,每加完一个样品,应更换一个加样头,以防污染,加样时勿碰坏样品孔周围的凝胶面.(注意:加样前要先记下加样的顺序). 4. 电泳:加样后的凝胶板立即通电进行电泳,电压60-100V,样品由负极(黑色)向正极(红色)方向移动.电压升高,琼脂糖凝胶的有效分离范围降低.当溴酚蓝移动到距离胶板下沿约1cm处时,停止电泳. (5)电泳完毕后,取出凝胶,用含有0.5 ug/ml的溴化乙锭1×TAE溶液染色约20 min,再用清水漂洗10 min. (6)观察照相:在紫外灯下观察,DNA存在则显示出红色荧光条带,采用凝胶成像系统拍照保存. 四,常见问题及注意事项 1.配琼脂糖时应使其完全熔化后方可制胶. 2.琼脂糖凝胶易于破碎,操作时要轻缓. 3.电泳时应注意电源线路,预防触电. 4.溴化乙淀具有致癌作用,配制及使用时应带乳胶或一次性塑料手套.并在专门的实验室内使用. 5.紫外线对人体有损伤作用,开灯时间不宜太长,注意防护. 6.DNA带形状模糊:DNA加样过多;电压太高;凝胶中有气泡. 7.质粒DNA的存在形式有3种,?共价闭环DNA(cccDNA),常以超螺旋形式存在;?开环DNA(ocDNA),此种质粒DNA的两条链中有一条发生一处或多处断裂,因此可以自由旋转从而消除张力,形成松弛的环状分子;?线状DNA,因质粒DNA的两条链在同一处断裂而造成.因此质粒DNA电泳的结果中有可能出现三条泳带, 5 它们的泳动速度为:cccDNA > 线状DNA > ocDNA. 添加来自TIANGEN的资料: 1,琼脂糖:不同厂家,不同批号的琼脂糖，其杂质含量不同,影响DNA的迁移及其荧光背景的强度,应有选择的使用. 2,凝胶的制备:凝胶中所加的缓冲液应该与电泳槽中的相一致,溶化的凝胶应该及时倒入板中,避免倒入之前凝固结块,倒入板中的凝胶应该避免出现气泡,以免影响电泳结果. 3,电泳缓冲液:为保持电泳所需的离子强度和PH,应经常更新电泳缓冲液. 4,样品加入量:一般情况下,0.5CM宽的梳子可加0.5微克的DNA量,加样量的多少依据加样孔的大小及DNA中片断的数量和大小而定.当加样孔大时,样品上样量应相应加大,否则会造成条带浅甚至辨认不清楚;反之,则应该适当减少加样量,但是上样量过多会造成加样孔超载,从而导致拖尾或扩散,对于较大的DNA此现象更明显. 5,DNA样品中盐浓度会影响DNA的迁移率,平行对照样品中应该使用同样的缓冲条件以消除这种影响. 6,DNA迁移率取决于琼脂糖的浓度,迁移分子的形状及其大小.采用不同浓度的凝胶有可能分辨范围广泛的DNA分子,制备琼脂糖凝胶可根据DNA分子的范围来决定凝胶的浓度.小片断DNA的检测应采用聚丙烯酰胺凝胶电泳,以提高分辨率. 什么是琼脂糖电泳的,其基本知识及操作方法是什么,不同形态的DNA在琼脂糖凝胶中迁移速率的大小如何分布或者改变, 什么是琼脂糖电泳的,其基本知识及操作方法是什么,不同形态的DNA在琼脂糖凝胶中迁移速率的大小如何分布或者改变, 以琼脂糖凝胶作为支持介质的电泳称为琼脂糖凝胶电泳。 二、琼脂糖凝胶电泳的准备和操作方法 1 、制备琼脂糖凝胶时注意避免产生气泡 凝胶电泳分为垂直型和水平型，一般垂直型分离效果好于水平型。但水平型可制备低浓度琼脂糖，制胶和加样比较方便，且电泳槽制作简单、价格低廉。 用缓冲液配制所需浓度的琼脂糖凝胶，水浴或微波炉中加热使琼脂糖完全融化，将溶液冷至 55 ? ，用少量琼脂糖凝胶将玻璃板或有机玻璃板封边，放置样品梳(梳齿下端离玻璃板 0.5 -1.0mm )。接着将融化的琼脂糖凝胶溶液不间断地倒在玻璃板上或有机玻璃板模子中(厚度依样品浓度而定，须注意避免气泡混入) , 室温下自然凝固。待完全凝固后小心取出加样器，在电泳槽中加入适量电泳缓冲液，使胶板浸没在电泳缓冲液下约 1mm 处。 2 、样品制备与加样 所加样品应有适当浓度与较小体积，用微量注射器缓慢加入样品，点样时电源必须处于关闭状态。为了指示电泳的距离，避免样品在电泳槽缓冲液中飘浮， 6 常在制备好的样品中加入含有蔗糖或甘油以及指示剂(溴酚蓝、二甲苯青等)的上样缓冲液。另外，样品中含盐量不能太高，否则电泳中会出现区带消失、前沿不齐等现象。样品中 DNA 含量以每条区带的 DNA 不少于 0.1 μ g 为宜， DNA 浓度过高会使电泳区带变宽，泳动距离异常。 样品的用量由加样孔的体积决定，通常为 10-50 μ g 。 3 、电泳 电泳温度视需要而定。对于大分子的分离，宜低温，电压应低(一般不超过 5V/cm )。普通电泳可在室温进行，电泳的电压为 5-15V/cm 。 4 、染色,以 DNA 电泳为例 常用荧光染料溴化乙锭( EB )进行染色以观察在琼脂糖凝胶中的 DNA 条带。 EB 能插入 DNA 分子中碱基对之间，使 EB 与 DNA 结合(超螺旋 DNA 与 EB 结合能力小于双链闭环，而双链闭环的 DNA 与 EB 结合能力小于线状双链 DNA )。将以染色的凝胶浸泡在 1mmol/lMgSO 4 溶液中 1 小时，可以降低未结合的 EB 所引起的背景荧光，以提高检测的灵敏度。 琼脂糖凝胶电泳对核酸的分离作用主要是依据它们的相对分子量质量及分子构型，同时与凝胶的浓度也有密切关系。 1、核酸分子大小与琼脂糖浓度的关系 (1)DNA分子的大小 在凝胶中，DNA片段迁移距离(迁移率)与碱基对的对数成反比，因此通过已知大小的标准物移动的距离与未知片段的移动距离时行比较，便可测出未知片段的大小。但是当DNA分子大小超过20kb时，普通琼脂糖凝胶就很难将它们分开。此时电泳的迁移率不再依赖于分子大小，因此，就用琼脂糖凝胶电泳分离DNA时，分子大小不宜超过此值。 (2)琼脂脂糖的浓度 如下表所示，不同大小的DNA需要用不同浓度的琼脂糖凝胶进行电泳分离。 表 琼脂糖浓度与DNA分离范围 琼脂糖浓度 /% 0.3 0.6 0.7 0.9 1.2 1.5 2.0 线状DNA大小/kb 60-5 20-1 10-0.8 7-0.5 6-0.4 4-0.2 3-0.1 2、核酸构型与琼脂糖凝胶电泳分离的关系 不同构型DNA的移动速度次序为:供价闭环DNA(covalently closed circular，cccDNA)>直线DNA>开环的双链环状DNA。当琼脂糖浓度太高时，环状DNA(一般为球形)不能进入胶中，相对迁移率为0(Rm=0)，而同等大小的直线双链DNA(刚性棒状)则可以长轴方向前进(Rm>0)，由此可见，这三种构型的相对迁移率主要取决于凝胶浓度，但同时，也受到电流强度、缓冲液离子强度等的影响。 生化及分子生物学试剂 >> λDNA/ Hind? Marker(单切Marker) 7 λDNA/ Hind? Marker*概述:λDNA/ Hind? Marker是由λDNA经Hind ?完全酶切并经灭活处理制成，浓度为 0.5μg/μL。已含有1xLoading Buffer,可以直接电泳。由8条片断组成:125bp、564bp、2027bp、2322bp、4361bp、6557bp、9416bp和23130bp。上样量0.5,1μg。 *储存:,20?保存一年以上，4?保存2,3个 Glycerol 5, 溴酚兰 0.02, 二甲苯腈蓝 0.02% 1×Loading Buffer说明: 1×loading Buffer中含有溴酚兰(Bromophenol Blue) 、二甲苯腈蓝FF (Xylene Cyanol FF)两种色素，用以判断DNA片断的电泳情况。溴酚蓝在琼脂糖凝胶中迁移速率是二甲苯腈蓝FF的2.2倍，这一特性与琼脂糖浓度无关。在0.5XTBE琼脂糖凝胶电泳中溴酚蓝迁移速率约与长300bp的线状双链DNA相同，而二甲苯腈蓝FF约与长4000bp的DNA相同。上述关系在浓度范围 0.5,,1.4,的琼脂糖凝胶中基本不受浓度变化的影响。 *注意事项 8 1( 电泳时电压不应超过20V/cm，电压过高，会导致电泳缓冲液发热从而影响DNA电泳效果。另外，DNA分子泳动过快，小片段DNA分子可能会跑出凝胶，而导致DNA片段丢失。 2( 对于λDNA cos位点，该位点退火之后，λDNA/ Hind? Marker的4361bp片段以及λDNA/ Hind?，EcoR ? Marker的3530bp片段可能全部或部分消失，为避免由于片段退火而产生DNA代带型的变化，应在点样前放在65?水浴中加热10min，然后在冰浴中冷却5min。 3( 一般情况下，0.5cm宽的梳子加样量为0.5μg的DNA量，即本产品点样量 5 μl左右。当加样孔大时，样品上样量应相应加大，否则会造成条带浅甚至辨认不清楚，反之则应当减少加样量，但是上样过多会造成片段脱尾或弥散，特别是对于较大DNA片段尤为明显。 4( DNA Marker中的小片段可能会在长时间电泳之后条带变得很弱，应选择合适电泳时间，或者加大EB浓度。 5(在DNA Marker的储存或使用过程中应避免反复冻融，长期冷冻保存过程中，可能会形成DNA浓度梯度，使用时应在解冻后进行振荡混合并进行短暂离心。 琼脂糖凝胶电泳实验技巧 作者:刘水平，罗志勇 来源:实用预防医学 时间:2008-5-19 摘要: 琼脂糖凝胶电泳是核酸分析常用的实验技术。虽然很多参考书上均有该方法的介绍El’ ，但实际操作中的一些技巧是无法从书本上获得的，本文介绍r琼脂糖凝胶电泳的实验技巧，这些经验源自多年的研究生教学实践，对初涉分子生物学实验者有一定的指导意义。 关键词: 琼脂糖凝胶;电泳;技巧 9 实用预防医学2006年8月 第13卷第4期Practical Preventive Medicine，Aug(2006，Vol 13，No 4 核酸分子是两性解离分子，在高于其等电点的电泳缓冲液中，其碱基不解离，而磷酸基团全部解离，核酸分子因而带负电荷，电泳时向正极迁移。琼脂糖主要从海洋植物琼脂中提取而来并经糖基化修饰，为一种聚合链线性分子，使用琼脂糖凝胶作为电泳支持介质，发挥分子筛功能，使得大小和构象不同的核酸分子的迁移率出现较大差异，从而达到分离的目的。琼脂糖凝胶电泳操作简单、快速，通过调整其使用浓度，使得分辨率达到大多数实验的要求，因此成为分离、鉴定、纯化核酸分子的常用方法。但操作过程中仍有不少要注意的问题。 1 凝胶制作 8, ,2(0,之间，如果一1(1 凝胶浓度 配制凝胶的浓度据实验需要而变 ，一般在0( 次配制凝胶100 ml，没用完的凝胶可以再次融化，但随着融化次数的增加，水分丢失也越多，凝胶浓度则会越来越高，导致实验结果不稳定，补水办法:一是在容器上标记煮胶前的刻度，煮胶后补充相应的水分至原刻度;二是在煮胶前称重，煮胶后补充水至原重量。粗略一点的方法是通过多次较恒定的煮胶条件得出一个经验补水值。以保证凝胶浓度基本维持在原浓度。核酸染色剂溴化乙锭(ethidium bromide)可加在融化的琼脂糖中，终浓度为0(5 t*g,ml;也可在电泳结束后染色。 1、2 梳板的选用 一般每个制胶模具均配有多个齿型不同的梳板，梳齿宽厚，形成的点样孑L容积较大，用于DNA 片段回收实验等;相反，梳齿窄而薄，形成的点样孑L容积就较小，用于PCR产物、酶切产物鉴定等。梳板的选择主要是看上样量的多少而定，一般来说，上样量小时尽量选择薄的梳板制胶，此时电泳条带致密清晰，便于结果分析。另外，每次制胶时都要注意梳齿与底板的距离至少要1 mm，否则，拔梳板时易损坏凝胶孑L底层，导致点样后样品渗漏。当然，点样孑L的破坏还与拔梳板的时间和方法有关，一般凝胶需冷却30 min以上方可拔梳板，应急的情况下可以将成型的凝胶块放4? 冰箱中冷却15 min 左右，拔梳板的方法是将制胶槽放置在电泳槽中的电泳缓冲液中，然后垂直向上慢慢用力，因为有液体的润滑作用，梳板易拔出且不易损坏点样孑L。 2 点样 点样需加上样缓冲液，因为上样缓冲液中加了甘油或蔗糖增加密度，使样品沉入孑L底; 10 指示样品的迁移过程，上样缓冲液中一般加了两种指示剂，溴酚兰和二甲苯青(值得注意的是指示剂并非染色剂，DNA染色剂是溴化乙锭，而且要在紫外光的激发下才能看见桔红色荧光)。上样缓冲液储存液一般为6× (10×)，表示其浓度为工作浓度的六倍。使用时上样缓冲液应稀释到一倍浓度。点样方法是将移液器基本垂直点样孑L，用另一只手帮助固定移液器下端，移液器枪头(Tip)尖端进入点样孑L即可将样品注入孑L内，千万不可将Tip尖插至孑L底，并点上适合的DNA分子量标准，所谓适合是指样品DNA分子量大小应基本在DNA分子量标准范围之内。 3 电泳 将电泳仪的正极与电泳槽的正极相连，负极与负极相连，核酸带负电荷，从负极向正极移动。电泳槽中电泳缓冲液与制胶用电泳缓冲液应相同，电泳缓冲液刚好没过凝胶1 mm 为好，电泳缓冲液太多则电流加大，凝胶发热。电泳时凝胶上所加电压一般不超过5 v,cm(指的是正负电极之间的距离，而不是凝胶的长度)，电泳时间一般为3O,60 min，根据实验需要也可作适当调整，电压增高，电泳时间缩短，核酸条带相对来说不够整齐，不够清晰;相反，电压降低，电泳时间较长，核酸条带整齐清晰。另外，如果电泳后样品泳动很慢或者没泳动，请检查胶模两端的封口胶条是否已去掉。 4 结果分析 较成功的电泳结果是分子量标准条带整齐清晰，样品条带也整齐清晰，如果条带模糊暗淡，单从琼脂糖凝胶电泳角度来说，可能的原因:溴化乙锭的质和量怎样?溴化乙锭见光易分解，母液配制时间过长或保存不当(一般4? 避光保存一年内有效)，或者终浓度没达到0(5 vg,ml;电泳槽中缓冲液使用次数过多，缓冲能力下降。特别是TAE缓冲液，一般用2,3次就要更换，TBE缓冲液则可使用10次左右。 实际工作中经常发现DNA 分子量标准小片段模糊不清，那足因为琼脂糖凝胶浓度一般不会超过2 0, ，较小的核酸片段 在它的分辨范围之内，并且EB带正电荷，电泳时会向负傲移动，如果将凝胶置含EB(0(5#g,m1)的水溶液中30 min，较小的片段则可重新染色:另外，溴化乙锭(EB)是一种中等强度诱变剂，操作过程中要戴手套，并将加有EB的染色液作好标记、妥善保存 。。 11 DNA电泳常见问题分析: 电泳出现拖尾现象 电泳出现拖尾现象，英文成为smear，就是弥散。 琼脂糖凝胶电泳拖尾是怎么回事, 1:样品浓度过高。这种情况稀释一下就行了。提质粒的时候常出现这种情况。 2:蛋白杂质阻碍DNA的泳动。提基因组DNA时常出现这种情况。 以上两种情况，拖尾都是在电泳条带的后面。 12 3:样品破碎或是被降解 4:存在RNA 这两种情况，拖尾都是在电泳条带的前面。 PCR产物电泳拖尾，主要从以下两个方面考虑: 1、PCR产物自身原因: 往往由于酶量过多或酶的质量差，dNTP浓度过高，Mg2+浓度过高，退火温度过低，循环次数过多，而造成PCR的非特异性产物过多。 对策:?减少Taq酶的量，或调换另一来源的酶。?减少dNTP的浓度。?适当降低Mg2+浓度。?增加量，减少引物的用量，减少循环次数，提高退火温度。 2、电泳体系的问题: (1)电泳缓冲液TAE或者TBE的污染，建议更换缓冲液。(2)上样时样品漂了，建议增加上样缓冲液的用量，以及小心加样。(3)电压太高。(4)适当把你的胶的浓度加大。(根据你的片断大小而定)(5)观察你的marker是否也存在拖尾现象，作为对照。 PCR拖尾及假阳性的原因及对策 拖尾 现象:产物在凝胶上呈Smear状态。 原因: 1.模板不纯 2.Buffer不合适 3.退火温度偏低 13 4.酶量过多 5.dNTP、Mg2+浓度偏高 6.循环次数过多 对策: 1.纯化模板 2.更换Buffer 3.适当提高退火温度 4.适量用酶 5.适当降低dNTP和镁离子的浓度 6.减少循环次数 假阳性 现象:空白对照出现目的扩增产物 原因: 靶序列或扩增产物 的交*污染 对策: 1.操作时应小心轻柔，防止将靶序列吸入加样枪内或溅出离心管外; 2.除酶及不能耐高温的物质外，所有试剂或器材均应高压消毒。所用 离心管 及加样枪头等均应一次性使用。 3.各种试剂最好先进行分装，然后低温贮存 14 (英文版 ) Two regulations promulgated for implementation is in the party in power for a long time and the rule of law conditions, the implementation of comprehensive strictly strategic plan, implementation in accordance with the rules and discipline to manage the party, strengthen inner-party supervision of major initiatives. The two regulations supporting each other, the < code > adhere to a positive advocate, focusing on morality is of Party members and Party leading cadres can see, enough to get a high standard; < rule > around the party discipline, disciplinary ruler requirements, listed as "negative list, focusing on vertical gauge, draw the party organizations and Party members do not touch the" bottom line ". Here, the main from four square face two party rules of interpretation: the first part introduces two party Revised regulations the necessity and the revision process; the second part is the interpretation of the two fundamental principles of the revision of laws and regulations in the party; the third part introduces two party regulations modified the main changes and needs to grasp several key problems; the fourth part on how to grasp the implementation of the two regulations of the party. < code > and < Regulations > revised the necessity and revised history of the CPC Central Committee the amendment to the Chinese Communist Party members and leading cadres honest politics several guidelines > and < Chinese Communist Party discipline and Punishment Regulations > column 1 by 2015 to strengthen 15 party laws and regulations focus. Two party regulations revision work lasted a Years, pooling the wisdom of the whole party, ideological consensus, draw historical experience, respect for the wisdom of our predecessors, which reflects the unity of inheritance and innovation; follow the correct direction, grasp the limited goals, adhere to the party's leadership, to solve the masses of the people reflect a focus on the problem. The new revision of the < code > and < rule >, reflects the party's 18 and the eighth session of the third, the spirit of the fourth plenary session, reflecting the experience of studying and implementing the General Secretary Xi Jinping series of important speech, reflects the party's eighteen years comprehensive strictly practice. (a) revised two regulations of the party need of < the ICAC guidelines > in < in 1997 Leaders as members of the Communist Party of China clean politics certain criteria (Trial) > based on revised, the promulgation and implementation of January 2010, to strengthen the construction of the contingent of leading cadres play an important role. But with the party to manage the party strictly administering the deepening, has not been able to fully meet the actual needs. Content is too complicated, "eight prohibition, 52 are not allowed to" hard to remember, and also difficult to put into practice; the second is concisely positive advocated by the lack of prohibited provisions excessive, no autonomy requirements; the third is banned terms and discipline law, both with the party discipline, disciplinary regulations 16 repeat and Criminal law and other laws and regulations repeat; the fourth is to "clean" the theme is not prominent, not for the existing problems, and is narrow, only needle of county-level leading cadres above. < rule > is in 1997 < Chinese Communist Party disciplinary cases (Trial) > based on revision, in December 2003 the promulgation and implementation, to strengthen the construction of the party play very important role. Along with the development of the situation, which many provisions have been unable to fully meet the comprehensive strictly administering the practice needs. One is Ji law, more than half of the provisions and criminal law and other countries laws and regulations Repetition; two is the political discipline regulations is not prominent, not specific, for violation of the party constitution, damage the authority of Party Constitution of misconduct lack necessary and serious responsibility to pursue; third is the main discipline for the leading cadres, does not cover all Party members. Based on the above situation, need to < the criterion of a clean and honest administration > and < rule > the two is likely to be more relevant regulations first amendment. By revising, really put the authority of Party discipline, the seriousness in the party tree and call up the majority of Party members and cadres of the party constitution of party compasses party consciousness. (II) two party regulations revision process the Central Committee of the Communist Party of China attaches great importance to two regulations revision . Xi Jinping, general books recorded in the Fifth 17 Plenary Session of the eighth session of the Central Commission for Discipline Inspection, on the revised regulations < > made clear instructions. According to the central deployment, the Central Commission for Discipline Inspection from 2014 under six months begin study two regulations revision. The Standing Committee of the Central Commission for Discipline Inspection 4 review revised. Comrade Wang Qishan 14 times held a special meeting to study two regulations revision, amendment clarifies the direction, major issues of principle, path and target, respectively held a forum will listen to part of the province (area) secretary of the Party committee, Secretary of the Discipline Inspection Commission, part of the central ministries and state organs Department The first party committee is mainly responsible for people, views of experts and scholars and grassroots party organizations and Party members. Approved by the Central Committee of the Communist Party of China, on 7 September 2015, the general office of the Central Committee of the Party issued a notice to solicit the provinces (autonomous regions, municipalities) Party, the central ministries and commissions, state ministries and commissions of the Party (party), the General Political Department of the military, every 3 people organization of Party of two regulations revision opinion. Central Commission for Discipline Inspection of extensive solicitation of opinions, careful study, attracting, formed a revised sent reviewers. In October 8 and October 12, Central Committee Political 18 Bureau Standing Committee and the Political Bureau of the Central Committee After consideration of the two regulations revised draft. On October 18, the Central Committee of the Communist Party of China formally issued two regulations. Can say, two laws amendment concentrated the wisdom of the whole party, embodies the party. Second, < code > and < Regulations > revision of the basic principles of two party regulations revision work and implement the party's eighteen, ten eight plenary, the spirit of the Fourth Plenary Session of the Eleventh Central Committee and General Secretary Xi Jinping important instructions on the revised < low political criterion > and < Regulations >, highlighting the ruling party characteristics, serious discipline, the discipline quite in front of the law, based on the current, a long-term, advance as a whole, with Bu Xiuding independent < rule > and < rule >. Main principle is: first, adhere to the party constitution to follow. The constitution about discipline and self-discipline required specific, awaken the party constitution of party compasses party consciousness, maintaining the authority of the constitution. General Secretary Xi Jinping pointed out that "no rules, no side round. Party constitution is the fundamental law, the party must follow the general rules. In early 2015 held the eighth session of the Central Commission for Discipline Inspection Fifth Plenary Session of the 16th Central Committee, Xi Jinping again pointed out that constitution is the party must follow the general rules, but also the general rules." the revision 19 of the < code > and < rule > is Method in adhere to the regulations established for the purpose of combining rule of virtue is to adhere to the party constitution as a fundamental to follow, the constitution authority set up, wake up the party constitution and party rules the sense of discipline, the party constitution about discipline and self-discipline specific requirements. 4 second is to adhere to in accordance with the regulations governing the party and the party. The Party of rule of virtue "de", mainly refers to the party's ideals and beliefs, excellent traditional style. The revised the < code > closely linked to the "self-discipline", insisting on the positive initiative, for all members, highlight the "vital few", emphasized self-discipline, focusing on the morality, and the majority of Party members and the ideological and moral standards. The revised < > Ji method separately, Ji, Ji Yan to Method, as a "negative list", emphasizing the heteronomy, focusing on vertical gauge. Is this one high and one low, a positive reaction, the strict party discipline and practice results transformation for the integration of the whole party to observe moral and discipline requirements, for the majority of Party members and cadres provides benchmarking and ruler. Third, insist on to. In view of the problems existing in the party at the present stage, the main problems of Party members and cadres in the aspect of self-discipline and abide by the discipline to make clearly defined, especially the party's eighteen years strict political discipline and political rules, organization and discipline and 20 to implement the central eight provisions of the spirit against the four winds and other requirements into Disciplinary provisions. Not one pace reachs the designated position, focusing on in line with reality, pragmatic and effective. After the revision of major changes, major changes in the < code > and < rule > modified and needs to grasp several key problems (a) < code > < code > adhere to according to regulations governing the party and party with morals in combination, for at the present stage, the leadership of the party members and cadres and Party members in existing main problems of self-discipline, put forward principles, requirements and specifications, showing Communists noble moral pursuit, reflected at all times and in all over the world ethics from high from low 5 common requirements. One is closely linked to the "self-discipline", removal and no direct relation to the provisions of . the second is adhere to a positive advocate, "eight prohibition" 52 are not allowed to "about the content of the" negative list moved into synchronization amendment < cases >. Three is for all the party members, will apply object from the leadership of the party members and cadres to expand to all Party members, fully embodies the comprehensive strictly required. The fourth is prominent key minority, seize the leadership of the party members and cadres is the key, and put forward higher requirements than the ordinary Party members. Five is to simplify, and strive to achieve concise, easy to understand, easy to remember. The revised < code > is the ruling Party since the first insists on 21 a positive advocate forAll Party members and the self-discipline norms, moral declaration issued to all members of the party and the National People's solemn commitment. > < criterion of a clean and honest administration consists of 4 parts, 18, more than 3600 words. After the revision of the < code >, a total of eight, 281 words, including lead, specification and Party member cadre clean fingered self-discipline norms, etc. Part 3 members low-cost clean and self-discipline, the main contents can be summarized as "four must" "eight code". Lead part, reiterated on ideal and faith, fundamental purpose, the fine traditions and work style, noble sentiments, such as "four must" the principle of requirements, strong tone of self-discipline, The higher request for 6 and supervised tenet, the foothold in permanent Bao the party's advanced nature and purity, to reflect the revised standards requirements. Members of self-discipline norms around the party members how to correctly treat and deal with the "public and private", "cheap and rot" thrifty and extravagance "bitter music", put forward the "four norms". Party leader cadre clean fingered self-discipline norms for the leadership of the party members and cadres of the "vital few", around the "clean politics", from civil servant of the color, the exercise of power, moral integrity, a good family tradition and other aspects of the leadership of the party members and cadres of the "four norms" < > < norm norm. "The Party member's self-discipline norms" and "party members and leading cadre clean fingered self-discipline norms," a 22 total of eight, collectively referred to as the "eight". "Four must" and "eight" of the content from the party constitution and Party's several generation of leaders, especially Xi Jinping, general secretary of the important discussion, refer to the "three discipline and eight points for attention" statements, and reference some embody the Chinese nation excellent traditional culture essence of epigrams. (2) the revised regulations, the main changes in the revised Regulations > to fully adapt to the strictly requirements, reflects the according to the regulations governing the law of recognition of deepening, the realization of the discipline construction and Jin Ju. < rule > is party a ruler, members of the basic line and follow. And the majority of Party members and cadres of Party organizations at all levels should adhere to the bottom line of thinking, fear discipline, hold the bottom line, as a preventive measure, to keep the party's advanced nature and purity. 1, respect for the constitution, refinement and discipline. Revised < rule > from comprehensive comb physical constitution began, the party constitution and other regulations of the Party of Party organizations and Party discipline requirements refinement, clearly defined in violation of the party constitution will be in accordance with regulations to give the corresponding disciplinary action. The original 10 categories of misconduct, integration specification for political discipline, discipline, honesty and discipline masses Ji Law and discipline and discipline and other six categories, the content of < rule > 23 real return to Party discipline, for the majority of Party members and listed a "negative list. 7 2, highlighting the political discipline and political rules. > < Regulations according to the stage of the discipline of outstanding performance, emphasizing political discipline and political rules, organization and discipline, in opposition to the party's leadership and the party's basic theory, basic line, basic program and basic experience, the basic requirement of behavior made prescribed punishment, increase the cliques, against the organization such as violation of the provisions, to ensure that the central government decrees and the Party of centralized and unified. 3, adhere to strict discipline in the law and discipline In front, Ji separated. Revised < Regulations > adhere to the problem oriented, do Ji separated. Any national law existing content, will not repeat the provisions, the total removal of 79 and criminal law, repeat the content of the public security management punishment law, and other laws and regulations. In the general reiterated that party organizations and Party members must conscientiously accept the party's discipline, die van comply with national laws and regulations; at the same time, to investigate violations of Party members and even criminal behavior of Party discipline and responsibility, > < Regulations distinguish five different conditions, with special provisions were made provisions, so as to realize the connection of Party discipline and state law. 4, reflect Wind building and anti-corruption struggle of the latest achievements. < rule > the party's eighteen years 24 implement the spirit of the central provisions of the eight, against the requirements of the "four winds" and transformation for disciplinary provisions, reflecting the style construction is always on the road, not a gust of wind. In the fight against corruption out of new problems, increase the trading rights, the use of authority relatives profit and other disciplinary terms. Prominent discipline of the masses, the new against the interests of the masses and ignore the demands of the masses and other disciplinary terms and make provisions of the disposition and the destruction of the party's close ties with the masses. Discipline to protect the party's purpose. 8 of these regulations, a total of three series, Chapter 15, 178, more than 24000 words, after the revision of the regulations a total of 3 series, Chapter 11, 133, 17000 words, divided into "general" and "special provisions" and "Supplementary Provisions" Part 3. Among them, add, delete, modify the provisions of the proportion of up to nearly 90%. 1, the general general is divided into five chapters. The first chapter to the regulations of the guiding ideology, principles and scope of application of the provisions, highlight the strengthening of the party constitution consciousness, maintenance the authority of Party Constitution, increase the party organizations and Party members must abide by the party constitution, Yan Centralized centralized, would examine at all levels of the amended provisions implementing and maintaining Party discipline, and consciously accept the party discipline, 25 exemplary compliance with national laws and regulations. The second chapter of discipline concept, disciplinary action types and effects of the regulations, will be a serious warning from the original a year for a year and a half; increase the Party Congress representative, by leaving the party above (including leave probation) punishment, the party organization should be terminated its representative qualification provisions. The third chapter of the disciplinary rules of use prescribed in the discipline rectifying process, non convergence, not close hand classified as severely or heavier punishment. "Discipline straighten "At least eighteen years of five years, these five years is to pay close attention to the provisions of the central eight implementation and anti -" four winds ". The fourth chapter on suspicion of illegal party disciplinary distinguish five different conditions, with special provisions were made provisions, to achieve effective convergence of Party and country 9 method. < rule > the provisions of Article 27, Party organizations in the disciplinary review found that party members have committed embezzlement, bribery, dereliction of duty dereliction of duty and other criminal law act is suspected of committing a crime shall give cancel party posts, probation or expelled from the party. The second is < Regulations > Article 28 the provisions of Party organizations in the disciplinary review But found that party members are stipulated in the criminal law, although not involved in a crime shall be investigated for Party discipline and responsibility should 26 be depending on the specific circumstances shall be given a warning until expelled punishment. This situation and a difference is that the former regulation behavior has been suspected of a crime, the feeling is quite strict, and the latter for the behavior not involving crime, only the objective performance of the provisions of the criminal code of behavior, but the plot is a crime to slightly. < Regulations > the 29 provisions, Party organizations in the discipline review found that party members and other illegal behavior, affect the party's image, the damage to the party, the state and the people's interests, we should depend on the situation Seriousness given disciplinary action. The loss of Party members, seriously damaging the party's image of behavior, should be given expelled from the party. At this article is party member is in violation of the criminal law outside the other illegal acts, such as violates the public security administration punishment law, customs law, financial laws and regulations behavior. The fourth is < cases > Article 32 stipulates, minor party members and the circumstances of the crime, the people's Procuratorate shall make a decision not to initiate a prosecution, or the people's court shall make a conviction and exempted from criminal punishment shall be given within the party is removed from his post, probation or expelled from the party. Party members and crime, sheets were fined in accordance with For acts; the principal Ordinance amended the provisions of the preceding paragraph. This is the new content, in order to achieve Ji method effective 27 convergence. Five is < > the thirty third article 10 of the provisions, the Party member due to an intentional crime is sentenced to criminal law (including probation) sheets or additional deprivation of political rights; due to negligence crime and was sentenced to three years or more (excluding three years) a penalty, shall give expelled punishment. Due to negligence crime is convicted and sentenced to three years (including three years) in prison or be sentenced to public surveillance, detention, shall in general be expelled from the party. For the individual may not be expelled from the party, should control Approval. This is followed and retained the original > < Regulations the provisions of punishment party authorization rules and report to a level party organizations. For is "party members with criminal acts, and by the criminal punishment, generally should be expelled from the party". The fifth chapter of probationary Party member of the discipline and discipline after missing members of the treatment and punishment decisions, such as the implementation of the provisions, clear the related party discipline and punishment decision made after, for duties, wages and other relevant alteration formalities for the longest time. 2, sub sub section will the original regulations of 10 categories of acts of violation of discipline integration revised into 6 categories, respectively, in violation of the punishments for acts of political discipline "in violation of discipline behavior of punishment" in violation of integrity of disciplinary action points "of violation punishments for acts 28 of mass discipline" "the violation of work discipline, punishment" in violation of discipline of life behavior punishment "6 chapters. 3, annex" Supplementary Provisions "clear authority making supplementary provisions of, cases of interpretative organ, as well as regulations implementation time and retroactivity etc.. 11 (3) learning understanding > < regulations needs to grasp several key problems The first problem -- about the violation of political discipline behavior > < new ordinance chapter 6 the political discipline column for the six disciplines, that is the main opposition to Party leadership and the opposition of the basic theory, basic line, basic program and basic experience, basic requirements of misconduct made provisions of the disposition, especially the eighteen since the CPC Central Committee put forward the Yan Mingzheng treatment of discipline and political rules requirements and practical achievements transformation for Discipline article, increase the false debate central policies, cliques, against the organization review, make no discipline of the principle of harmony terms. These are the party's eighteen years in comprehensive strictly Process combined with the practice of rich content. (1) false debate the central policies and undermine the Party of centralized and unified the problem is made in accordance with the provisions of the party constitution. Constitution in general programme requirements adhere to democratic centralism is one of the requirements of the construction of the party must adhere to the four cardinal. Application 29 of this principle is not only the party the basic organization principle and is also the mass line in party life, it requires that we must fully develop inner-party democracy, respect for the dominant position of Party members, safeguarding the Party member democratic rights, give full play to the enthusiasm and creativity of the party organizations at all levels and Party members, at the same time, also must implement the right concentration, ensure the party's mission < the chaos in unity and concerted action to ensure that the party's decision to get quickly and effectively implementing. The Party Central Committee formulated the major principles and policies, through different channels and ways, fully listen to the party organizations and Party members of the opinions and suggestions, but 12 is some people face to face not to say back blather "" will not say, after the meeting said, "" Taiwan does not say, and nonsense ", in fact, not only disrupt the people thought, some causing serious consequences, the damage to the Party of the centralized and unified, hinder the central policy implementation, but also a serious violation of the democratic system of principles. There is no doubt that shall, in accordance with the Regulations > 4 Specified in Article 6 to give the appropriate punishment. For did not cause serious consequences, to give criticism and education or the corresponding tissue processing. (2) about the destruction of the party's unity < New Regulations > the forty eighth to fifty second article, to damage Party's unity unified and violation of political discipline, punishment situation 30 made explicit provisions. Article 52 of the new "in the party get round group, gangs seek private gain, cliques, cultivate private forces or through the exchange of interests, for their own to create momentum and other activities to gain political capital, given a serious warning or withdraw from their party posts disposition; if the circumstances are serious, to give Leave a party to observation or expelled from the party. (3) on against the organization review of the provisions of the constitution, party loyalty honesty is party members must comply with the obligations. Members must obey the organization decision, shall not violate the organization decided encounters by asking questions to find organization, rely on the organization, shall not deceive the organization, against the organization. For example, after the investigation does not take the initiative to explain the situation, but to engage in offensive and defensive alliance, hiding the stolen money is against survey organization, is a violation of the behavior of political discipline. Article 24 of the original > < Regulations, although the provisions of the interference, hinder group review the behavior of the fabric can be severely or 13 Aggravated punishment, but did not put this kind of behavior alone as a discipline for qualitative amount of discipline. > < new regulations increase the Article 57, "anti organization review, one of the following acts, given a warning or serious warning; if the circumstances are relatively serious, giving removed from or placed on probation within the party post; if the circumstances are serious, give 31 expelled from the party: (a) on supply or forged, destroyed, transfer, conceal evidence; (II) to prevent others expose, providing evidence Material; (III) harboring co personnel; (4) to the organization to provide false information, to hide the fact; (5) the him against the acts of the organization review. "< rule > add this clause to the Constitution requires more specific, the previous no punishment in accordance with the definite list and put forward clear punishment in accordance with. (4) about organizing or participating in superstitious activities as < Regulations > about engage in activities of feudal superstition obstruction of social management order" violations of Article 164 the provisions, but according to the original < rule > only in disrupting production, work, social life order The case to be disciplinary treatment, in other words, alone make the feudal superstition, organize or participate in the activities of feudal superstition of, does not constitute a violation. Which is not consistent with the requirements of our party's political party. > < new regulations in this change is, superstitious activities on the political discipline, increase the Article 58, is the organization of Party members, in superstitious activities included in violation of the negative list of political discipline deserves punishment, which and Party members should adhere to the correct political principle, political standpoint and viewpoint is consistent. 14 the second question about organization and discipline violation behavior of democratic centralism is our The party's fundamental 32 organizational system. < New Regulations > Chapter 7 "in violation of the behavior of organizational discipline punishment" mainly for violation of democratic centralism, contrary to the "four obey the discipline behavior for source classification rules. Increased not in accordance with the relevant provisions or requirements to the organization for instructions to report on major issues; do not report truthfully report about personal matters; falsify personal archives; hide before joining the party serious mistakes; leading cadres in violation of the relevant provisions of the organization, will participate in the spontaneous formation of the old Xiang, Alumni Association, comrades in arms; to obtain illegal country residence abroad or foreign nationality, illegal for private frontier Documents such as discipline terms. (1) on the report truthfully report personal matters in 2010 the Central Committee of the Communist Party of China office, office of the State Council introduced < provisions on issues related to the leading cadres to report personal >, is clear about the request, deputy division level and above leading cadres should truthfully report changes in my marriage and spouse, children, moved to the country (territory), practitioners, income, real estate, investment and other matters, for failing to report, do not report, concealed and unreported, according to the seriousness of the case, giving criticism and education, and to make a correction within a time limit, shall be ordered to make a check, the commandment Jiemian conversation, informed criticism or jobs, free Post 33 processing, constitute violations, in accordance with the relevant provisions shall be given a disciplinary sanction. But since the original < Regulations > and there is no corresponding specific terms, the violation of the regulations, do not report, as a false report about personal matters, there is no corresponding disciplinary action terms, in practice it is difficult to operate. In this regard, in order to solve is not reported, as a false report about personal matters 15 asked censure, the new < Regulations > add the Article 67 of violation of personal matters related to reporting requirements, report truthfully report the clear punishment basis, making this kind of violation behavior is no longer free drilling for Exhibition on matters of personal checks to verify and supervision of cadres, discipline review provides a powerful discipline guarantee. (2) about the illegal organization, to fellow, alumni, friends of the war will < regulations stipulated in Article 68 of >, leading cadres of the party in violation of relevant provisions of the organization, will participate in the spontaneous formation of fellow, Alumni Association, comrade in arms to give the punishment according to the seriousness of the case. Here special needs note is three points: this provision for only the leading cadres ", reflecting the high requirements of leading cadres; second violation in 2002, the Central Commission for Discipline Inspection, the central Organization Department and the The relevant provisions of the General Political Department jointly issued the < off in leading cadres not to participate in 34 the initiative to set up "the villagers would be" alumni "" comrades in arms organization notice >. That is to say, to the spontaneous formation of fellow, Alumni Association, comrades in arms will constitute the premise of discipline is a violation of the provisions of this. The notification specified, leading cadres are not allowed to participate in the spontaneous incorporation of fellow, alumni, between comrades association would like the organization and shall not bear the sorority Human and the organizer shall not hold the corresponding position in the sorority; shall not borrow machine woven "network" and engage in kiss sparsely, round and round the gangs, but not "align" "Jieyi Gold Orchid" behavior. The third is to emphasize here that shall be organized to participate in the initiative to set up the villager, Alumni Association, comrades in arms. The so-called 16 spontaneous was established mainly means without registration. Therefore, Party members include leading cadres in the normal range of fellow, alumni, comrades in the party is not a violation of the rules of Party discipline. The three problem about integrity violations discipline Lian Jie discipline has been eighteen years clean government and anti The focus of the work of corruption. This Ordinance to amend, honesty and discipline this biggest adjustment, the new content, most of which prescribed by the original < criterion of a clean and honest administration > 8 ban and 52 are not allowed into the basic this part. (1) the central eight provisions of the spirit and requirements into < New > in 35 the regulations of the party's eighteen years, central resolutely implement the provisions of the eight and pay close attention to the node and intensive briefing, on public funds, private bus, public funds tourism, gifts of public funds, the big parade and lavish weddings and festive, illegal payment allowances and subsidies, illegal construction of buildings such as the original masses reflects the relatively strong, "four winds" problem of the Resolute rectification. But original < Regulations > to eat and drink, super standard reception and no clear and specific expression, new < rule > will implement eight Central provisions of the spirit of the problem increased to "clean cheap self-discipline" chapter, clear to exceed the standard, beyond the scope of the reception or borrow machine eating and drinking etc. some in violation of the provisions of the spirit of the eight central relevant persons responsible for punishment, once the violation will be according to the < rule > severely punished, binding, enhanced significantly. One is to add Article 87, about to obtain illegal, hold, and the actual use of the sports card, golf ball cards and other consumer card. Illegal access to private clubs made the punishment provisions. The second is increase the Article 97, has made provisions of the disposition of illegal self pay or 17 spamming allowances, subsidies and bonuses. The third is added to the Article 99, on violation of super standard, beyond the scope of the reception or borrow machine eat and drink to make the provisions of punishment. The fourth is to increase the Article 101, on the management 36 of conference activities in violation of the provisions made sanctions regulations. The fifth is the increased Article 102, for violation of office space management provisions made the punishment provisions. Six is to split the original < rule > Article 78, formed article 98, Article 100 of public Models of tourism, violate the provisions on the administration of the use of discipline of the bus through the list of made a more detailed provisions, apply more operational. Seven is increased the article 96, in violation of the relevant provisions of the, to participate in public funds to pay for dinner, high consumption of entertainment, fitness activities and public funds to buy gift, send gifts to make the provisions of the disposition. On the violation of the central provisions of the spirit of the eight, in addition to > < new regulations into "violation of honesty and self-discipline" misconduct outside, is different with the original < Regulations >, revised < rule > clearly defined not only to disposition of the directly responsible persons, but also dispose of collar Guide responsibilities. (2) increased trading rights, use of authority or position influence as relatives and close to profit violation of the terms of the < New Regulations > absorption < clean politics several guidelines > the relevant provisions spirit, increase the "negative list, including trading rights, the functions and powers or duties of influence as relatives and staff around profit. Increase Article 81" mutual use of office or authority ring for each other and their spouses, children and their spouses and other relatives, 37 around 18 personnel and other specific relationship between the people to seek benefits engage in trading rights, given a warning or serious warning Sanctions; if the circumstances are relatively serious, giving removed from or placed on probation within the party post; if the circumstances are serious, shall be expelled from the party. Increase Article 82 "connivance, acquiescence to the spouse, children and spouse etc. relatives and staff around the effect of Party members and cadres personal authority or position for personal gain, if the circumstances are relatively minor, given a warning or a serious warning; if the circumstances are relatively serious, giving removed from or placed on probation within the party post; if the circumstances are serious, the given Expelled from the party. Party members and cadres of the spouses, children and their spouses does the actual work and get salary or although the actual work but to receive significantly beyond the same rank salary standard, Party members and cadres informed fails to correct, in accordance with the provisions of the preceding paragraph. "(3) about the gifts, gifts misconduct from handling practices in recent years, engaged in official cadres received gifts, gifts problems more prominent. This has seriously affected the image of Party members and cadres, damaged the relations between the party and the masses, and is a hotbed of corruption, is really necessary for this kind of behavior to be disciplined. < rule > no on accepting gifts, gifts, Card consumption behavior to engage in simple "one 38 size fits all", but the difference between the different separately. One is in accordance with the provisions of Article 83, accepting may affect the impartial enforcement of the official gifts, gifts, consumer cards to depending on the seriousness of the case shall be given disciplinary sanctions. That is to say, for may affect the fair execution of business gifts, gifts, consumer cards are not allowed to accept. 19 is in accordance with the provisions of Article 83, accepting was significantly higher than normal ceremony is still exchanges of gifts, gifts, consumer card to disciplinary action. This is new regulations that daily life is purely reciprocity, accepting the same thing, boys , fellow friends gifts, gifts, shopping cards, although and fair execution of business has nothing to do, also want to as the case shall be dealt with, the situation is obviously beyond normal reciprocity ". The so-called" reciprocity ", one is emphasizes the reciprocal in protocol. In other words is you to me how, I to you how, not only does not go. The second is to significantly exceeded the normal local economy level, customs and habits, economic capacity of individual gifts, gift value. Specific sanctions to according to the processing of a variety of factors to consider, as appropriate. The third is the root according to the provisions of Article 84," to Engaged in public service personnel and their spouses and children, children's spouses and other relatives and other specific relationship presented Ming Xianchao normal reciprocity of gifts, gifts, consumer card, if the circumstances are 39 relatively serious, given a warning until probation. "In accordance with the above provisions, gift giving significantly beyond the normal reciprocity, giver constitute the discipline. (4) on illegal trading of stocks or of other regulations of securities investment < > Article 88 is engaged in punishment regulation of camp and activity in violation of the relevant provisions on. Among them, 3 will" buying and selling stocks or in other securities investment "column as one of disciplinary cases Out. First need to be clear, > < regulations did not change the provisions of the Ordinance. The ordinance of the provisions of the second paragraph of Article 77 provisions "who, in violation of the provisions of the sale of stocks" is one of the violation, and at the beginning of the first paragraph of this article "violation of 20 against the relevant provisions of the" textual representation of the repeat revision. In, just from the legislative technique removed the "personal" in violation of the provisions, content and not to change the original provisions. Need to clear is, "buy or sell a stock or of other securities investment" refers to violation of the relevant provisions refers to in April 2001, the Party Central Committee, the State Council promulgated the On the work of the party and government organs staff's personal investment in securities if dry provisions >. According to the provisions of Article 3, use of work time, office facilities, the sale of stocks and securities investment fund belongs to illegal behavior. The fourth question, about the violation of the masses of the disciplinary actions < 40 new regulations > would violate public discipline behavior of a single set of a class of restored "three rules of discipline and eight note" in the discipline of the masses of excellent traditional. < New Regulations > Chapter 9 "to violate the masses of disciplinary action", mainly the destruction of the party's close ties with the masses of misconduct made provisions on disciplinary; enrich and perfect the super standard, beyond the scope of Xiang Qun < All to raise fund and labor, in for involving the public affairs deliberately, chinakayao, in social security, policy support, disaster relief funds and materials and other matters assigned any kind and affectionate friends, obviously unfair acts against the interests of the masses of the disciplinary terms; increase the terms not in accordance with the provisions of the public party provided, government, factory and village (neighborhood) provided etc. violation of the people's right to know the behavior of the discipline. The fifth issue, on the violation of work discipline violation of work discipline added "negative list, including Party organizations carry out strictly Main responsibility for poor discipline terms. (1) the new party organization comprehensive strictly the main responsibility of the party does not fulfill the 21 or perform poor disciplinary terms < New Regulations > has a lead people to pay attention to the new terms, that is, in chapter ten "on the violation of work discipline behavior punishment" increase in the one hundred and fourteenth "party fail to perform 41 comprehensive strictly the main responsibility of the party or to perform comprehensive strictly the main responsibility for administering the ineffective, resulting in serious damage or serious adverse effects, to the person directly responsible and the responsibility of leadership and give a warning or serious warning. The situation is serious, giving removed from their party posts or on probation Punishment. "Here the changes. For the first time, the main responsibility write < rule >. (2) new shall report does not report or false reporting discipline shall increase the 117 bar" in the higher level units to check to inspect the work or report to the parent unit, work report of shall report the matters do not report or do not report, cause serious damage or serious adverse effects of, to the person directly responsible and the responsibility of leadership, to give warning or a serious warning; if the circumstances are serious, giving removed from their party posts or placed on probation. "For example, during the tour visits to the region, the unit Members the obligation to the inspection teams to reflect the true situation, to conceal not reported or intentionally to the inspection teams to provide false information, causing serious damage or serious adverse effects of, the person directly responsible and the responsibility of leadership should be in accordance with the provisions of < rule > Article 117 to give the appropriate punishment. (3) the new not granted in accordance with the provisions of punishment, not according to the provisions of the implementation of disciplinary measures 22 violation 42 of the provisions of Article 115 "Party organizations have one of the following circumstances, the direct leadership responsibility. Probation: members were sentenced punished, in accordance with the provisions give disciplinary action or in violation of state law Rules of behavior should be given disciplinary punishment and dispose of; disciplinary punishment by decision or complaint review the decision made, not in accordance with the provisions of the implementation decisions on punishment of the party, position, rank, and the treatment of the matters; Party members subject to disciplinary action, not in accordance with the cadre management authority and organizational relationships of by dispose of Party members to carry out education and supervision of daily management. "What needs to be pointed out is," cliff "demoted with cars, housing, secretary problem, implementation is not a discipline and party posts and ranks treatment by the organization department is responsible for the implementation, with cars, housing by the logistics department is responsible for the implementation of, Ji Commission responsible oversight responsibilities. The six questions, about the acts violating the discipline of life "in violation of discipline of life behavior punishment", mainly to "four winds" problems and a serious violation of social morality, family virtue acts of misconduct made provisions on disciplinary, an increase of extravagant life, contrary to the social public order and good customs disciplinary terms. Worth mentioning is, > < Regulations in Article 150 43 about "adultery" "mistress (Cardiff) formulation in the new < rule > is removed, to expand the scope of Article 127 provisions" and others hair improper sexual relations, let face disciplinary action Wider more strict. Four, and earnestly do a good job in the < code > and < rule > implement system of life lies in execution. < code > and < Regulations > is 23 Party organizations at all levels of a strong constraint and all Party members follow. Learning and implementing the < code > and < rule > is party committees (party), commission for discipline, discipline inspection group), the majority of Party members and cadres of the common responsibility, must party caught together, the party one execution. () Party committees (party) and play and implement comprehensive strictly the main responsibility, pays special attention to the two party regulations learning cross penetration. One is to Adhere to the party constitution is fundamental to follow, and resolutely safeguard the authority of the Party Chapter. The second is to adhere to the problem oriented, to strictly political discipline and political rule moments in the first place. The third is to adhere to the discipline and rules quite in front must not allow the bottom line to break the discipline. The fourth is to seriously organize the study of propaganda and education, in the city party members to create a disciplined, about the rules of the strong atmosphere. (II) Commission for Discipline Inspection at all levels (Discipline Inspection Group) to an important basis for the revised two party regulations as, adhere to easily 44 blame, strengthen supervision and enforcement And early method in combining Ji, increase accountability efforts. One is to two regulations within the party important basis, adhere to the party constitution duties, with strict discipline to maintain the constitution authority. The second is to adhere to high standards and keep the bottom line, adhere to Ji Yan in law and discipline, the discipline and rules quite in front, grasping grasping small, easily blame, comprehensive use of supervision and discipline of "four kinds of form" in the discipline embodies the strict requirements and care for the discipline, tighten up, Yan. According to discipline in accordance with regulations to carry out disciplinary review. Increase the violations punishment, clues can initiate an investigation on the trial of cases According to the discipline on the provisions of the proportion of processing and control manager maintains discipline. To focus on investigating non convergence, don't accept hand serious disciplinary violations at the same time, pay more attention to the general review of discipline 24 behavior, and gradually increase the lighter punishment. Four is to illuminate two party regulations, combined with the local district of the Department of party conduct Lianzheng to relevant laws and regulations system clean, timely research proposes to establish, change, waste release opinion, avoid conflict, and the fasten system of cage, and gradually formed not rot, not rot, not want to rot system and working mechanism. (3) the leadership of the party members and cadres 45 should play an exemplary role. The rate of higher consciousness of the front of the Ordinance to lead practice self-discipline norms, leading to maintain discipline of seriousness and authority. One is the above rate, demonstration and guidance, take the lead in learning and mastering the < code > and < > the requirements and regulations, do the deep understanding, learning to use. The second is to play an exemplary role in abiding by discipline rate, take the lead in practicing self-discipline norms, firmly establish before the discipline are equal, the system has no privilege, discipline is no exception, and consciously do standards, require more strict measures more practical. Third is to consciously accept supervision, conscientiously participate in the democratic life and debriefing honesty responsibility, such as Real to the party organization to report personal matters, and to accept the supervision of the work and life of the normal, habits under the supervision of the exercise of power, to carry out the work. The fourth is to set an example for the strict enforcement of discipline. Leading cadres should take the lead in the maintenance of discipline of seriousness and authority, to dare to seriously, dare to struggle, dare to offend people, for misconduct not laissez faire, but do not indulge, not tolerate to ensure the party constitution of party compasses party put in place. (4) the majority of Party members and cadres to set high standards and hold the bottom line, consciously abide by the < code > and < rule >. One is to control the < standard > and < 25 cases >, tight Close connection 46 with their own thoughts, practical work and life, efforts to solve the problems, to enhance the study and implement of the effectiveness and pertinence. The second is to establish a high standard and hold the bottom line, consciously in the self-discipline pursuit of high standards, strictly in the party away from the red line discipline, discipline, about the rules and know fear, distrust, the formation of the honor system, comply with the system, safeguard system of good habits. Regulations and discipline regulations of < < New Revision of the Communist Party of China clean fingered self-discipline criterion > and < Chinese Communist Party Disciplinary Regulations > is the Party Central Committee in the new situation to promote the comprehensive strictly root of the lift, the regulations of the party construction of keeping pace with the times. We should study and implement the Standards > and < > as the primary political task, adhere to the party constitution duties, adhere to the discipline of, Ji Yan in law, the full implementation of supervision and discipline accountability responsibilities for coordination and promotion "four overall" strategic layout in XX District vivid practice to provide a strong guarantee. - Si Jian Wu, and effectively enhance the implementation of the implementation of the < code > < > The consciousness of General Secretary Xi pointed out that the party is in charge of the party, to manage the party; strictly, it can cure good party. Criterion > and < rule > keep pace 47 with the times is our party to form a new potential strictly rules, to deepen understanding of the ruling law, is a comprehensive strictly, and strengthen the supervision within the party important grasper. Criterion > closely linked to the theme of self-discipline, self-discipline intensity-modulated, focusing on morality, for the majority of Party members and cadres set to see, feel the high standard, show the Communists noble moral sentiment; District Ordinance < < rule > the constitution of discipline integration into a political discipline, organization and discipline, honesty and discipline, discipline of the masses, work discipline, discipline, emphasize discipline, focusing on established rules, to draw the party organizations and Party members do not touch the bottom line. Party committees (party) to effectively strengthen the study and publicize the implementation of the < code > and < rule > the organization and leadership of criterion > and < > a full range, multi angle, deep publicity, the formation of study and publicize and implement the boom, in order to consolidate the development of good political environment to create a positive environment and atmosphere. The majority of Party members and cadres to strict requirements, the < rule > and < Ordinance cases > as a guide to action and behavior criterion, adhere to the moral standards and discipline of the bottom line, to maintain the discipline of worship and awe, learning, compliance, and maintain party discipline and rules of the model. The discipline inspection and supervision organs and cadres to the < code > and < > as the deepening of 48 the "three", and strengthen supervision and discipline of accountability is important to follow correctly grasp the use of supervision and discipline of "four types", promote the comprehensive strictly in new roots. Strict discipline, adhere to the discipline and rules quite in front of the enterprise in the implementation of expensive to implement to execute. The discipline inspection and supervision organs to implement the < code > and < Efforts to investigate cases > as an opportunity, adhere to the party should manage the party strictly, serious performance of their duties to promote two regulations implementation. Change discipline concept. From the simple "investigation of illegal" to a comprehensive "stare at violate discipline"; from the business case to prosecute the big cases and timely solve the signs of tilt to. From the orientation to the normal supervision; from the tube "minority" to resist the "most". Guide Party members and cadres to seriously implement the < code > and < rule >, consciously abide by the political, organizational, honest people, work, life six discipline. Urge the leadership cadres sent to play an exemplary role, take the lead in strictly enforced. Relaxation and rest Strong wind is Su Ji, closely linked to the "four winds" prone to multiple, the masses reflect the strong important nodes and, glued to the four winds "new form, new trends, seriously investigate and deal with violations of the central eight provisions of the spirit, to increase the bulletin exposure intensity, the formation of Chajiu" four winds "loudly, continue to release and discipline 49 must be strict signal. At the same time, extended supervision tentacles, through the development of the rural and fishery party conduct Lianzheng construction inspection, issued in a timely manner, Chajiu occurred in grassroots side of corruption and unhealthy, and earnestly safeguard the interests of the masses and social stability. The creativity and discipline. Grasp and make good use of the supervision and discipline of" four types ", to ensure the Promoting clues to lots of discipline and the law of the vacuum tube, strict together. Not only to the "less" and "very few" thorough investigation severely punished, but also the "most" to maintain zero tolerance trend through layers of conductive pressure, let the discipline about the rules for each party members and cadres consciously follow. At present, it is necessary to implement the "list" management, strictly follow the clues to the disposal of five standards, make the classification of disposal, dynamic cleanup. Zadok Party committees at all levels to implement the main responsibility, comprehensive use of disciplinary action and tissue processing and other means to effectively curb the commonplace, used to see do not blame the "small problem", not because of "practice" and "exception". Prominent Discipline characteristics, the problems of violation of discipline and rules into the focus of supervision and discipline and disciplinary review. At the same time, increase case double check the intensity, the serious problems of discipline violations or "four winds" ban but not absolutely, not only to be 50 held directly responsible, but also held leadership responsibility; not only to pursue studies the main responsibility of the Party committee, and to pursue Commission for Discipline Inspection and supervision responsibilities and promote accountability to become the new norm. The implementation of supervisory responsibility. Maintain < code > and < rule > the seriousness and authority of the discipline and rules and truly become the party's ruler. Discipline as supervision within the party the specialized agency, we must strengthen the responsibility to act, adhere to the Impartial discipline accountability, earnestly will be strict discipline, discipline must be punished, and severely punish the acts in violation of rules and regulations. 51